Department of Microbiology and Immunology, Graduate Program in Cancer Biology, Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, Miami, Florida 33136, USA.

Abstract

Upon virus infection, the innate immune response provides the first line of protection and rapidly induces type I interferons (IFNα/β), which mediate potent antiviral effects. To maintain homeostasis and prevent autoimmunity, IFN production is tightly regulated; however, the mechanisms of negative regulation are poorly understood. Herein, we demonstrate that the A20 binding inhibitor of NF-κB 1 (ABIN1) is a novel negative regulator of antiviral signaling. Overexpression of ABIN1 inhibited IFN-β promoter activation in response to virus infection or poly(I:C) transfection, whereas siRNA-mediated knockdown of ABIN1 enhanced IFN-β production upon virus infection. ABIN1 interacted with the A20 regulatory molecule TAX1BP1 and was essential for the recruitment of TAX1BP1 and A20 to the noncanonical IκB kinases TBK1 and IKKi in response to poly(I:C) transfection. ABIN1 and TAX1BP1 together disrupted the interactions between the E3 ubiquitin ligase TRAF3 and TBK1/IKKi to attenuate lysine 63-linked polyubiquitination of TBK1/IKKi. Finally, an intact ubiquitin binding domain of ABIN1 was essential for ABIN1 to interact with TBK1/IKKi and inhibit IFN-β production upon poly(I:C) transfection or virus infection. Together, these results suggest that ABIN1 requires its ubiquitin binding domain and cooperates with TAX1BP1 and A20 to restrict antiviral signaling.

ABIN1 inhibits TBK1/IKKi ubiquitination by disrupting interactions between TRAF3 and TBK1/IKKi.A, 293T cells were transfected with GFP-IKKi (left) or GFP-TBK1 (right) (1 μg of each). After 48 h, cells were harvested, and lysates were subjected to co-IP and immunoblotting (IB) with the indicated antibodies. B, wild-type MEFs were transfected with poly(I:C) (12 μg) where indicated, and cells were harvested 2 h later. Lysates were subjected to co-IP and immunoblotting with the indicated antibodies. C, Abin1−/− MEFs were transfected with GFP-ΔRIG-I (1 μg) and V5-ABIN1 (2 μg), as indicated. After 24 h, cells were harvested, and lysates were subjected to co-IP and immunoblotting with the indicated antibodies. D and E, 293T cells were transfected with HA-Lys-63 ubiquitin (500 ng), V5-ABIN1 (1 μg), FLAG-TBK1 (1 μg), or FLAG-IKKi (1 μg), as indicated. After 48 h, cells were harvested, and lysates were subjected to co-IP and immunoblotting with the indicated antibodies. F, 293T cells were transfected with GFP-TAX1BP1, V5-ABIN1, FLAG-IKKi (left), or FLAG-TBK1 (right) (1 μg of each), as indicated. After 48 h, cells were harvested, and lysates were subjected to co-IP and immunoblotting with the indicated antibodies. G, 293T cells were transfected with GFP-TAX1BP1 (1 μg), control siRNA, or ABIN1 siRNA (60 pmol), as indicated. After 48 h, cells were transfected with poly(I:C) (12 μg) and harvested after 4 h. Lysates were subjected to co-IP and immunoblotting with the indicated antibodies.

Model depicting the proposed role of ABIN1 in the inhibition of antiviral signaling. In response to viral infection, the RNA helicases RIG-I/MDA5 recognize viral nucleic acid and trigger a downstream signaling cascade, including the mitochondrial adaptor molecule IPS1 and the E3 ubiquitin ligase TRAF3. TRAF3 ubiquitinates the noncanonical IKK kinases TBK1/IKKi, which, when activated, phosphorylate IRF3, leading to its dimerization and nuclear translocation where it activates IFN-β transcription. ABIN1 is recruited to ubiquitinated forms of TBK1/IKKi via its UBD and also recruits TAX1BP1 and A20 to form an A20 regulatory complex. The A20 complex disrupts interactions between TBK1/IKKi and TRAF3 to attenuate antiviral signaling.